Page 1 of 22 The vaccinia virus A56 protein: a multifunctional ...

The vaccinia virus A56 protein: a multifunctional transmembrane glycoprotein that anchors 

two secreted viral proteins 

Brian C. DeHaven, 1 Kushol Gupta 2 and Stuart N. Isaacs 1,3 

1 Department of Medicine, Division of Infectious Diseases University of Pennsylvania School of 

Medicine, Philadelphia, PA 19104, USA 

2 Department Biochemistry & Biophysics and Howard Hughes Medical Institute, University of 

Pennsylvania School of Medicine, Philadelphia, PA 19104, USA 

3 Infectious Diseases Section, Philadelphia Veterans Affairs Medical Center, Philadelphia, PA 

19104, USA 

JGV Papers in Press. Published June 29, 2011 as doi:10.1099/vir.0.030460-0 

Correspondence 

Stuart N. Isaacs 

isaacs@mail.med.upenn.edu 

Page 1 of 22

The vaccinia virus A56 protein was one of the earliest-described poxvirus proteins 

with an identifiable activity. While originally characterized as a haemagglutinin 

protein, A56 has other functions as well. A56 is capable of binding two viral 

proteins, a serine protease inhibitor (K2) and the vaccinia virus complement 

control protein (VCP), and anchoring them to the surface of infected cells. This is 

important; while both proteins have biologically relevant functions at the cell 

surface, neither one can locate there on its own. The A56–K2 complex reduces the 

amount of virus superinfecting an infected cell and also prevents the formation of 

syncytia by infected cells; the A56–VCP complex can protect infected cells from 

complement attack. Deletion of the A56R gene results in varying effects on 

vaccinia virus virulence. In addition, since the gene encoding the A56 protein is 

non-essential, it can be used as an insertion point for foreign genes and has been 

deleted in some viruses that are in clinical development as oncolytic agents. 


Introduction – the A56 protein 

Orthopoxviruses are some of the most complex viruses infecting humans and include variola virus, 

the causative agent of smallpox, and vaccinia virus, which is used as a live vaccine. Although 

smallpox has been eradicated, vaccinia virus (VACV) is still studied as a model organism to 

understand basic aspects of pox virology, as a vaccine vector for immunizations against other 

infectious agents and for oncolytic cancer therapy. It is also studied because human infections with 

zoonotic poxviruses like monkeypox virus still occur. While VACV is a large virus, containing a 

genome of nearly 200 kbp that encodes more than 200 proteins, the genome is still small compared 

with that of the host cell. For this reason, VACV encodes a number of multi-functional proteins, 

one of which is A56. The A56 protein is able to bind two other viral proteins, a serine protease 

inhibitor (K2) and the vaccinia virus complement-control protein (VCP), and express them at the 

surface of the infected cell. The A56–K2 complex binds to the entry–fusion machinery of VACV; 

this reduces superinfection and prevents cell–cell fusion of infected cells. The A56–VCP complex 

protects infected cells from complement attack and contributes to viral virulence. These complexes 

were only discovered recently, but the history of A56 protein studies extends much farther back in 

time. 

The A56 protein was one of the first VACV genes to be identified and studied (Nagler, 1942). This 

was because what is now called the A56 protein had haemagglutination activity; it was called 

VACV haemagglutinin (HA) from the 1940s until near the end of the 20th century. The presence of 

a VACV protein with HA activity was believed to be important because several other viruses such 

as influenza, measles and mumps viruses contained proteins with HA activity. However, unlike 

these other viral envelope proteins, which were important for viral entry, a biologically relevant 

function could not ultimately be ascribed to the VACV HA activity. Nevertheless, the HA activity 

of poxviruses allowed the classification of poxviruses into those that had HA activity and those that 

did not. Members of the orthopoxvirus genus were the only ones that had HA activity (Fenner et al., 

1988). Furthermore, haemabsorption and HA-inhibition assays were early methods used to 

differentiate between various orthopoxviruses and variola virus isolates from various parts of the 

world (Fenner et al., 1988). Later, when the protein (now called ‘A56’) and the gene (designated 

‘A56R’ but also designated VACWR181 in VACV strain WR) responsible for this activity was first 

identified (Ichihashi, 1977; Shida, 1986a), the A56R ORF was used to build phylogenetic trees that 

showed the genetic relationships between the members of the genus Orthopoxvirus (Hutin et al., 

2001). 

The ability to track the protein by its haemagglutinating activity also provided a means to show that 

the protein is found on the cell membrane of the infected cell (Blackman & Bubel, 1972), but not on 

mature virus (MV), the most abundant form of virus made during an infection (Fig. 1). While the 

protein is not found on MV, it is present on another infectious form of virus called the extracellular 


virus (EV) (Payne & Norrby, 1976). The EV is critical for the efficient spread of the virus in vitro 

and in vivo. However, unlike the other glycoproteins found on EV, the A56 protein has considerably 

more sequence variability between different orthopoxviruses. This may be because, unlike the other 

EV glycoproteins, A56 is not essential for formation of the EV. As mentioned earlier, this sequence 

variability, initially recognized in HA-inhibition assays, made A56R a useful ORF for building 

phylogenetic trees. 

Based on the A56 protein sequence, the calculated molecular mass of the protein is ~35 kDa, but 

the protein contains putative sites for both N- and O-glycosylation and runs at an apparent 

molecular mass of 85 kDa (Brown et al., 1991; Payne, 1992; Shida & Dales, 1981). Thus, A56 is 

heavily glycosylated and this glycosylation is linked to its HA activity. The positions of the five 

predicted N-linked glycosylation sites are shown in Fig. 2. Blocking N-linked glycosylation with 

tunicamycin results in an apparent molecular mass of 62 kDa and this moiety retains its HA 

activity. In the presence of tunicamycin, A56 is still able to incorporate labelled glucosamine, 

confirming that the protein also undergoes O-linked glycosylation. Because HA activity is lost 

when the protein is completely deglycosylated, this implies that O-linked glycans are involved in 

the HA activity (Shida & Dales, 1981). A prior review on A56 by Hisatoshi Shida (Shida, 1989) 

pointed out that A56 was the first viral protein shown to be O-glycosylated, and that this protein 

provided additional evidence that O-linked glycosylation occurs as a protein transits through the 

Golgi apparatus (Shida & Dales, 1981; Spiro, 1966). There are predicted to be as many as 23 Olinked 

glycosylation sites, which all fall between residues 149 and 255 (with 20 falling between 

residues 175 and 230). The heavy glycosylation of the A56 protein may be the reason why anti-A56 

antibodies cannot neutralize EV or protect from infection (Galmiche et al., 1999; Pulford et al., 

2004). Interestingly, completely unglycosylated A56 protein migrates with an apparent molecular 

mass of 58 kDa; this is different from the 35 kDa calculated from the sequence of the ORF (Shida, 

1986b). Shida pointed out that this discrepancy is not because of other post-translational 

modifications because in vitro translation of the A56 ORF resulted in a protein that migrates at ~60 

kDa (Shida, 1989). Thus, this is a clear example of a difference between predicted and observed 

molecular masses, of the type that are known to occur. Additionally, the A56R gene has two 

separate promoters for early and late gene expression. Interestingly, late expression also produces a 

smaller, 68 kDa form of the protein (Brown et al., 1991), although the biological significance of this 

smaller protein is not known. Both the 85 and 68 kDa forms of the protein can be seen by using 

Western blotting at late time points. 

A schematic diagram of the A56 protein is shown in Fig. 2. The ~100 aa N-terminal region of A56 

(after the signal sequence) has sequence similarities with the immunoglobulin (Ig) superfamily. 

This region of the protein is more highly conserved among orthopoxviruses than the rest of the A56 

ectodomain (Aguado et al., 1992; Cavallaro & Esposito, 1992). In this Ig domain, it was 


hypothesized that the two cysteines in the ectodomain form an intramolecular disulfide bridge (Jin 

et al., 1989). Following on from this, modern computer modelling of this portion of the A56 protein 

supports formation of an Ig domain structure, shows the first two cysteines of A56 to be in close 

proximity and predicts that these cysteines form an intramolecular bond (Fig. 3). While not 

experimentally proven, but based on phenotypes of mutated viruses, the Ig domain may be involved 

in the observed cell–cell fusion regulatory activity. An analysis of VACV mutants produced by 

chemical mutagenesis (Shida & Matsumoto, 1983) revealed that the cell–cell fusion regulatory 

properties and haemabsorption properties of the A56 protein are separate (Seki et al., 1990). While 

A56R gene-knockout viruses cannot perform either function, one point mutant (Glu121 to Lys) was 

found to be HA negative but could still prevent syncytia formation. Since the exact mechanism of 

haemagglutination is not known, it is difficult to speculate as to why this mutation causes a loss of 

activity, especially when the O-linked glycosylation was linked to HA activity. Additionally, a virus 

containing a Cys103 to Tyr mutation did not have HA activity or the ability to prevent syncytia. As 

discussed earlier, this cysteine is predicted to form an intramolecular disulfide bond (Figs 2 and 3), 

so this mutation is likely to destabilize the entire domain, resulting in an abnormally folded protein 

that has been reported to make it to the cell surface, but which does not have HA activity or prevent 

syncytia formation (Seki et al., 1990). As discussed later, a third cysteine at position 162 (just 

before the first tandem repeat) has recently been shown to form a disulfide bridge with VCP 

(DeHaven et al., 2010). Between the Ig domain and the transmembrane domain there are two 

tandem-repeat motifs, which start near residue 170 and continue until residue 238 (Jin et al., 1989). 

These repeats, which are predicted to be heavily modified by O-glycosylation, do not share 

sequence similarity with other proteins. While the function of the tandem repeats in the A56 protein 

are unknown, tandem repeats are often important in protein structure and function. The 

transmembrane domain, which anchors the protein in membranes, has been implicated as an 

important domain that results in A56 interacting with the F13 protein (also called VP37) (Oie et al., 

1990). Since the F13 protein is a key protein in the formation of EV (Blasco & Moss, 1991), F13 

may help direct the incorporation of A56 into the EV envelope. A short intracellular domain of ~12 

aa that is present at the C terminus of the protein may be responsible for trafficking A56 protein out 

of the endoplasmic reticulum (ER) and into the Golgi apparatus (Shida & Matsumoto, 1983). 

Analysis of a panel of viruses with various HA and syncytia-inducing phenotypes revealed that an 

A56 protein lacking the cytoplasmic tail was transported from the ER to the Golgi apparatus more 

slowly but could still make it to the cytoplasmic membrane, while a protein with an aberrant 

cytoplasmic tail [owing to an upstream nucleotide insertion(s)] interfered with transport of the 

protein out of the ER. 


A56 protein inhibits spontaneous cell–cell fusion of VACV infected cells 

by forming a complex with K2 

The ability to haemagglutinate was not the only early function ascribed to A56. Cells infected with 

certain strains of VACV can result in cell–cell fusion, and in 1971 this was directly linked to a lack 

of A56 protein (Ichihashi & Dales, 1971). Interestingly, the loss of another viral protein, K2, also 

causes infected cells to fuse. This was reported by three different groups in 1992 (Law & Smith, 

1992; Turner & Moyer, 1992; Zhou et al., 1992). Recently, it has been shown that the A56 and K2 

proteins form a complex on the surface of infected cells, and that this complex is responsible for 

preventing syncytia formation (Turner & Moyer, 2006). The K2 protein is also found on EV 

particles, as observed by electron microscopy and proteomics studies (Brum et al., 2003; Manes et 

al., 2008). K2, also known as serine protease inhibitor (SPI)-3, is one of a family of poxvirus 

proteins that share sequence similarities with serpin proteins (Boursnell et al., 1988). K2 protein 

(SPI-3) has in vitro protease-inhibition activity (Turner et al., 2000; Wang et al., 2000), but this 

activity is not related to its ability to prevent syncytia formation (Turner & Moyer, 1995). While 

regions of K2 that are important for its interaction with A56 have been identified (Turner & Moyer, 

1995), the domain of A56 that interacts with K2 has not been identified. In a transient transfection 

system, A56 mutated at either Cys34 or Cys103 was able to be expressed on the cell surface (and 

bind VCP) (DeHaven et al., 2010) but could not bind K2 (B. C. DeHaven & S. N. Isaacs, 

unpublished), again implying that the Ig domain is involved in interacting with K2. 

The mechanism by which the A56/K2 complex inhibits spontaneous fusion of infected cells was not 

clear until the discovery that the virus encoded multi-subunit entry fusion complex (EFC), which 

was found on the MV membrane (Senkevich et al., 2005), interacted with the A56–K2 complex 

(Wagenaar & Moss, 2007). It was further shown that the A56–K2 complex interacts directly with 

the A16 and G9 proteins, two of the proteins that are part of the EFC (Wagenaar et al., 2008). This 

finding led to the hypothesis that the A56–K2 complex on infected cells could prevent reinfection 

of already infected cells (Moss, 2006). Support for this hypothesis was obtained experimentally 

when recombinant VACVs with deletions of one of the genes encoding the A56–K2 complex were 

used to infect cells, followed by superinfection with VACV carrying a luciferase reporter gene. 

Superinfection of cells that were infected by wild-type virus expressing the A56–K2 complex had 

significantly lower luciferase levels than cells infected with a virus expressing a mutated A56–K2 

complex (Turner & Moyer, 2008). It has also been shown that transfecting the A56R and K2L genes 

into cells is sufficient to diminish both infection and virion induced cell–cell fusion (Wagenaar & 

Moss, 2009). Antibodies against either the K2 (Turner & Moyer, 2006, 2008) or A56 proteins 

(Wagenaar & Moss, 2009) increase the amount of superinfection and cell fusion seen. This may be 

because of the antibodies either blocking the A56–K2 complex from interacting with the MV EFC 

or perhaps by causing the displacement of K2 from A56, rendering the complex non-functional. 


Since syncytia formation of infected cells is mediated by EV, it appears that the A56—K2 complex 

is inhibiting syncytia formation by ‘inhibiting’ EV re-entry into an infected cell. This would occur 

first by the non-fusogenic dissolution of the EV outer envelope (Law et al., 2006) that exposes the 

EFC machinery on the MV, and then entry into an already infected cell is inhibited by engagement 

with A56–K2. Many other viruses have evolved mechanisms to prevent superinfection prior to 

entry (Berngruber et al., 2010). For example, the influenza neuraminidase protein cleaves the entry 

receptor as new virus is released, preventing superinfection (Huang et al., 2008), while retroviruses 

can downregulate entry receptors (Lindwasser et al., 2007; Wildum et al., 2006). Other viruses, 

such as vesicular stomatitis virus, prevent superinfection by slowing endocytosis of new virus 

(Simon et al., 1990). Thus, while other viruses prevent superinfection prior to entry, none use a 

mechanism that actually interferes with their own entry proteins like orthopoxviruses. 

The A56–K2 complex on the surface of infected cells prevents superinfection by incoming MV 

particles. This should not be confused with the ability of another set of poxvirus proteins (A33 and 

A36) on infected cells that repel EV away from newly infected cells (Doceul et al., 2010). By 

repelling EV away from a newly infected cell, virus spread to distant uninfected cells is enhanced. 

Thus, VACV has evolved a way to prevent or at least reduce superinfection by MV and EV prior to 

virus entry. Given the vital role that EV plays in the spread of virus in vitro and in vivo, we 

postulate that the repulsion of EV away from newly infected cells has a more important function 

than the A56–K2 complex preventing superinfection by MV. However, the A56–K2 complex may 

play a valuable role early in the initial spread of virus away from the very first cells infected. 

The interaction of A56 and K2 has been shown to occur in VACV and cowpox virus (CPXV), and 

homologues of both proteins are present in all sequenced members of the genus Orthopoxvirus. 

There was a curious report of an ectromelia virus (ECTV) that caused syncytia formation despite 

the presence of the ECTV homologues of A56 and K2 (Erez et al., 2009). However, cell–cell fusion 

is a complicated process, and other factors may be at work. The phenotype described for this ECTV 

might be explained by the presence of mutations in other viral genes. Also of note is that Chang et 

al. have recently shown that MV surface proteins A25 and A26 are fusion suppressors (Chang et 

al., 2010). They found that mutated VACVs that contain deletions of these proteins cause 

spontaneous fusion at neutral pH. This may explain the syncytia-forming phenotype reported by 

Erez et al. (2009). 

Cell surface expression of VCP through interactions with A56 

K2 is not the only viral protein that directly interacts with the ectodomain of the A56 protein. 

Recently, a new interaction was discovered between A56 and VCP. VCP is a 35 kDa soluble 

protein that was previously characterized as being the major secreted protein from VACV-infected 

cells (Isaacs et al., 1992; Kotwal & Moss, 1988; Kotwal et al., 1990). VCP has the ability to inhibit 


multiple steps of the complement cascade (Bernet et al., 2004; Kotwal et al., 1990; Liszewski et al., 

2006, 2009; McKenzie et al., 1992; Miller et al., 1997; Mullick et al., 2005; Rosengard et al., 1999, 

2002; Sahu et al., 1998; Sfyroera et al., 2005). Research has shown that VCP-deletion viruses are 

mildly attenuated in vivo (DeHaven et al., 2010; Isaacs et al., 1992), possibly owing to an improved 

adaptive immune response in the absence of complement inhibition (Girgis et al., 2011). Besides 

being secreted, VCP was found to be expressed on the surface of infected cells (Girgis et al., 2008). 

Surface expression required the presence of the free N-terminal cysteine of VCP and the A56 

protein (Girgis et al., 2008). Furthermore, it was shown recently that the N-terminal free cysteine of 

VCP forms a covalent bond with the unpaired cysteine of the ectodomain of the A56 protein 

(Cys162) (DeHaven et al., 2010). This results in expression of VCP on the surface of infected cells. 

It is not yet known whether the K2 protein and VCP can interact with the same A56 molecule. 

Based on the domains of A56 that K2 and VCP interact with (Figs 2 and 3), it is possible that a 

heterotrimer could form. However, we favour a model where the A56 protein is expressed at early 

and late time points as a monomer and some A56 molecules interact with K2, while other molecules 

interact with VCP. We base this model on the temporal organization of VACV protein synthesis. 

The K2 protein is expressed from an early promoter (Turner & Moyer, 1995) while VCP is 

expressed from a late promoter (B. C. DeHaven & S. N. Isaacs, unpublished; Moulton et al., 2010). 

Thus, this would keep the K2 protein and VCP from competing for A56 molecules. 

In contrast to the apparently unique function of the A56–K2 complex for preventing superinfection 

by interacting with the viral EFC, surface expression of a complement-control protein is seen in 

other viruses. Several herpesviruses and flaviviruses express complement regulators that are found 

on the surface of infected cells (Bernet et al., 2003; Chung et al., 2006; Friedman et al., 1984; 

Harris et al., 1990). This A56–VCP complex can protect cells from complement-mediated lysis of 

infected cells, and surface-bound VCP may be important for full virulence in vivo (DeHaven et al., 

2010; Girgis et al., 2008). Proteomics data on MV and EV particles from VACV indicate that VCP 

is found on EV (Manes et al., 2008) (Fig. 1). While this has not been confirmed experimentally, it is 

intriguing to speculate that VCP trafficked to the EV envelope provides additional protection from 

the host complement attack. 

The interaction between VCP and A56 does not seem to be limited to VACV. The smallpox 

homologue of VCP, called SPICE (for smallpox inhibitor of complement enzymes), can also bind 

to the VACV A56 protein when plasmids expressing both proteins are transfected into cells. Also, 

immunofluorescence staining of ECTV-infected cells shows surface expression of its complementcontrol 

protein (called EMICE). Interestingly, in transfection studies, EMICE does not interact with 

the VACV A56 protein, but is able to bind to the ECTV A56 protein (DeHaven et al., 2010). This 

suggests co-evolution of these proteins might have occurred. It is unclear which protein might be 

driving this co-evolution, but, given the greater differences between orthopoxvirus A56 proteins in 


the domain where the complement control protein binds, we hypothesize that A56 may be driving 

this co-evolution. It is unclear whether there is evidence of similar co-evolution between A56 and 

K2. Despite only 83% identity between the CPXV and VACV A56 proteins, the CPXV K2 protein 

can interact with the VACV A56 protein and inhibit syncytia formation (Turner & Moyer, 1995). If 

the K2 protein interacts with the Ig domain of A56 this region has less sequence divergence 

(Aguado et al., 1992; Cavallaro & Esposito, 1992) and thus little co-evolution has been required. 

A56 and VACV virulence 

The A56 protein is not needed for viral replication in cell culture. However, the contributions of 

A56 to VACV virulence have not been fully elucidated and the data are inconsistent (see Table 1). 

While work with a plaque-purified virus (NYCBH strain) showed significant attenuation with an 

A56R gene-knockout virus in intracranial and intranasal challenge models (Lee et al., 1992), it was 

not compared to a gene-rescue virus and the knockout contained the �-galactosidase marker gene. 

Similarly, work with Western Reserve (WR)-strain-based VACV virus, in which the A56R gene 

was deleted, showed a small amount of attenuation when given by the intracranial route in young 

mice (Flexner et al., 1987). An additional study using WR showed an increase in LD50 levels in 

both intracranial and intraperitoneal models when A56 is knocked out (Shida et al., 1988). 

However, in this same study, no attenuation was seen when inserting a foreign gene [the human T- 

lymphotropic virus (HTLV) glycoprotein] in place of A56 in the LC16mO or LO strains; deleting 

A56 resulted in little-to-no attenuation in both intracranial and intraperitoneal models (Shida et al., 

1988). It should be noted that the LO and LC16mO strains are more attenuated than the WR strain. 

Based on these imperfect studies, it appears that deletion of the A56R gene does attenuate the virus 

in some cases, and this attenuation is more readily seen when starting with a virulent virus. 

Deletion of the VACV proteins that bind the A56 protein provides a clearer, if unfinished picture of 

the contribution of A56 to VACV virulence, as these mutants appear to result in different levels of 

virulence in vivo (Table 1). The deletion of the K2L ORF does not appear to markedly attenuate 

vaccinia. No differences were seen when comparing a wild-type VACV with a K2L-knockout 

VACV, which were given to mice by the intranasal route of infection (Law & Smith, 1992). 

Similarly, intranasal infection of mice with a K2L-knockout or wild-type CPXV showed nearly 

identical LD50 (Thompson et al., 1993). Since deletion of the K2L gene results in the same syncytia- 

producing phenotype as deletion of the A56R gene, these findings appear to indicate that the viruses 

that do not form a functional A56–K2 complex remain virulent in mice. These viruses may even 

compensate for the loss of superinfection control by increased pathogenesis because of syncytia 

formation. 

As previously mentioned, VCP is needed for full VACV (strain WR) virulence, as knockout viruses 

are attenuated in an intradermal rabbit model (Isaacs et al., 1992), and both intradermal and 


intranasal mouse models (DeHaven et al., 2010; Girgis et al., 2011). Importantly, a VCP mutant 

(VCPmut) that cannot form the A56–VCP complex (owing to mutation of the unpaired N-terminal 

cysteine of VCP) is also attenuated after intranasal and intradermal challenge (DeHaven et al., 

2010). Taken as a whole, these experiments seem to suggest that the A56–VCP complex is more 

important to virulence in mouse models than the A56–K2 complex. However, direct comparisons of 

mutations that affect these two complexes and appropriate rescue viruses have not been carried out. 

Gene screening using A56R-knockout viruses and oncolytic vectors 

Because the A56R gene is non-essential for virus replication, it has been used as a region of the 

VACV genome suitable for insertion and expression of foreign genes (Shida, 1989). The HA 

properties of the A56 protein allow for easy identification of recombinant plaques with foreign 

genes inserted into the A56R locus, even in the absence of a selection marker. That is, the addition 

of chicken erythrocytes to an infected cell monolayer will cause wild-type plaques to appear red, 

while recombinant viruses with A56 deleted will remain white (Oda, 1965; Shida & Matsumoto, 

1983). Another application of A56 mutants has been in the development of VACVs as oncolytic 

cancer vectors. Recently, a new vector was developed, GLV-1h68, which includes the deletion of 

A56 along with a series of other gene deletions [thymidine kinase (J2R) and F14.5L, a non-essential 

MV protein (Izmailyan & Chang, 2008)]. When compared with the parental virus where only J2R 

and F14.5 were deleted, the additional A56 knockout resulted in a virus that was further attenuated 

and able to reduce the size of breast cancer tumours in a nude mouse model. Why including an 

A56R gene deletion in this oncolytic virus improved the survival of tumour-bearing mice is 

unknown, but it is intriguing to speculate upon the potential contribution of infected tumour cells 

that did not express the K2 protein or VCP on their surfaces. GLV-1h68 has since been successfully 

used to shrink tumours in several xenograft mouse models (Gentschev et al., 2010; Lin et al., 2008; 

Worschech et al., 2009; Yu et al., 2009a, b). 

Conclusions 

The history of A56 is can be divided into two acts. In the first act, the protein was discovered and 

many basic characteristics about the protein were described. After a number of years of diminished 

work on A56, the last decade has seen a burst of studies that focus on the A56 protein. During this 

second act important discoveries about interactions with other viral proteins and their relevant 

biological functions have been made. The A56 protein is found in multiple locations during an 

infection (Fig. 1), and its interactions with other viral proteins produce multiple effects. While A56 

is a ‘non-essential’ protein, it is an important one: through its interaction with the K2 protein it is 

involved in preventing reinfection of already-infected cells, which may promote viral spread as well 

as inhibiting syncytia formation. Through its interactions with VCP, it is involved with defending 

infected cells from the immune response of the host. Research on A56 has also been incorporated 


into new oncolytic vectors, and new panels of mutant viruses are in progress that may assist in 

further elucidating the role A56 plays in pathogenesis. 

Acknowledgements 

The authors would like to thank Joe Esposito for helpful discussions and the reviewers of the 

submitted manuscript whose comments helped us improve this mini-review. S.N.I. is supported in 

part by Public Health Service grants U01-AI077913, U01-AI066333 and U54-AI057168 (Middle 

Atlantic Regional Center of Excellence in Biodefense and Emerging Infectious Diseases) from the 

National Institute of Allergy and Infectious Disease. P.C.D is supported by institutional training 

grants T32 AI07324 and U01-AI066333. 


References 

Aguado, B., Selmes, I. P. & Smith, G. L. (1992). Nucleotide sequence of 21.8 kbp of variola 

major virus strain Harvey and comparison with vaccinia virus. J Gen Virol 73, 2887–2902. 

doi:10.1099/0022-1317-73-11-2887 Medline 

Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. 

J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search 

programs. Nucleic Acids Res 25, 3389–3402. doi:10.1093/nar/25.17.3389 Medline 

Bernet, J., Mullick, J., Singh, A. K. & Sahu, A. (2003). Viral mimicry of the complement system. 

J Biosci 28, 249–264. doi:10.1007/BF02970145 Medline 

Bernet, J., Mullick, J., Panse, Y., Parab, P. B. & Sahu, A. (2004). Kinetic analysis of the 

interactions between vaccinia virus complement control protein and human complement 

proteins C3b and C4b. J Virol 78, 9446–9457. doi:10.1128/JVI.78.17.9446-9457.2004 

Medline 

Berngruber, T. W., Weissing, F. J. & Gandon, S. (2010). Inhibition of superinfection and the 

evolution of viral latency. J Virol 84, 10200–10208. doi:10.1128/JVI.00865-10 Medline 

Blackman, K. E. & Bubel, H. C. (1972). Origin of the vaccinia virus hemagglutinin. J Virol 9, 

290–296. Medline 

Blasco, R. & Moss, B. (1991). Extracellular vaccinia virus formation and cell-to-cell virus 

transmission are prevented by deletion of the gene encoding the 37,000-Dalton outer 

envelope protein. J Virol 65, 5910–5920. Medline 

Boursnell, M. E., Foulds, I. J., Campbell, J. I. & Binns, M. M. (1988). Non-essential genes in 

the vaccinia virus HindIII K fragment: a gene related to serine protease inhibitors and a gene 

related to the 37K vaccinia virus major envelope antigen. J Gen Virol 69, 2995–3003. 

doi:10.1099/0022-1317-69-12-2995 Medline 

Brown, C. K., Turner, P. C. & Moyer, R. W. (1991). Molecular characterization of the vaccinia 

virus hemagglutinin gene. J Virol 65, 3598–3606. Medline 

Brum, L. M., Turner, P. C., Devick, H., Baquero, M. T. & Moyer, R. W. (2003). Plasma 

membrane localization and fusion inhibitory activity of the cowpox virus serpin SPI-3 

require a functional signal sequence and the virus encoded hemagglutinin. Virology 306, 

289–302. doi:10.1016/S0042-6822(02)00017-X Medline 

Brünger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. 

W., Jiang, J. S., Kuszewski, J., Nilges, M. & other authors (1998). Crystallography & 


NMR system: A new software suite for macromolecular structure determination. Acta 

Crystallogr D Biol Crystallogr 54, 905–921. doi:10.1107/S0907444998003254 Medline 

Cavallaro, K. F. & Esposito, J. J. (1992). Sequences of the raccoon poxvirus hemagglutinin 

protein. Virology 190, 434–439. doi:10.1016/0042-6822(92)91229-N Medline 

Chang, S. J., Chang, Y. X., Izmailyan, R., Tang, Y. L. & Chang, W. (2010). Vaccinia virus A25 

and A26 proteins are fusion suppressors for mature virions and determine strain-specific 

virus entry pathways into HeLa, CHO-K1, and L cells. J Virol 84, 8422–8432. 

doi:10.1128/JVI.00599-10 Medline 

Chen, V. B., Arendall, W. B., III, Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. 

J., Murray, L. W., Richardson, J. S. & Richardson, D. C. (2010). MolProbity: all-atom 

structure validation for macromolecular crystallography. Acta Crystallogr D Biol 

Crystallogr 66, 12–21. doi:10.1107/S0907444909042073 Medline 

Chung, K. M., Liszewski, M. K., Nybakken, G., Davis, A. E., Townsend, R. R., Fremont, D. 

H., Atkinson, J. P. & Diamond, M. S. (2006). West Nile virus nonstructural protein NS1 

inhibits complement activation by binding the regulatory protein factor H. Proc Natl Acad 

Sci U S A 103, 19111–19116. doi:10.1073/pnas.0605668103 Medline 

DeHaven, B. C., Girgis, N. M., Xiao, Y., Hudson, P. N., Olson, V. A., Damon, I. K. & Isaacs, S. 

N. (2010). Poxvirus complement control proteins are expressed on the cell surface through 

an intermolecular disulfide bridge with the viral A56 protein. J Virol 84, 11245–11254. 


Doceul, V., Hollinshead, M., van der Linden, L. & Smith, G. L. (2010). Repulsion of 

superinfecting virions: a mechanism for rapid virus spread. Science 327, 873–876. 

doi:10.1126/science.1183173 Medline 

Erez, N., Paran, N., Maik-Rachline, G., Politi, B., Israely, T., Schnider, P., Fuchs, P., 

Melamed, S. & Lustig, S. (2009). Induction of cell-cell fusion by ectromelia virus is not 

inhibited by its fusion inhibitory complex. Virol J 6, 151. doi:10.1186/1743-422X-6-151 

Medline 

Eswar, N., Webb, B., Marti-Renom, M. A., Madhusudhan, M. S., Eramian, D., Shen, M. Y., 

Pieper, U. & Sali, A. (2006). Comparative protein structure modeling using Modeller. Curr 

Protoc Bioinformatics 5, Unit 5.6. Medline 

Fenner, F., Henderson, D. A., Arita, I., Jezek, Z. & Ladnyi, D. (1988). Smallpox and Its 

Eradication. Geneva: WHO. 


Flexner, C., Hügin, A. & Moss, B. (1987). Prevention of vaccinia virus infection in 

immunodeficient mice by vector-directed IL-2 expression. Nature 330, 259–262. 

doi:10.1038/330259a0 Medline 

Friedman, H. M., Cohen, G. H., Eisenberg, R. J., Seidel, C. A. & Cines, D. B. (1984). 

Glycoprotein C of herpes simplex virus 1 acts as a receptor for the C3b complement 

component on infected cells. Nature 309, 633–635. doi:10.1038/309633a0 Medline 

Galmiche, M. C., Goenaga, J., Wittek, R. & Rindisbacher, L. (1999). Neutralizing and 

protective antibodies directed against vaccinia virus envelope antigens. Virology 254, 71– 

80. doi:10.1006/viro.1998.9516 Medline 

Gentschev, I., Ehrig, K., Donat, U., Hess, M., Rudolph, S., Chen, N., Yu, Y. A., Zhang, Q., 

Bullerdiek, J. & other authors (2010). Significant growth inhibition of canine mammary 

carcinoma xenografts following treatment with oncolytic vaccinia virus GLV-1h68. J Oncol 

2010, 736907. Medline 

Girgis, N. M., Dehaven, B. C., Fan, X., Viner, K. M., Shamim, M. & Isaacs, S. N. (2008). Cell 

surface expression of the vaccinia virus complement control protein is mediated by 

interaction with the viral A56 protein and protects infected cells from complement attack. J 

Virol 82, 4205–4214. doi:10.1128/JVI.02426-07 Medline 

Girgis, N. M., Dehaven, B. C., Xiao, Y., Alexander, E., Viner, K. M. & Isaacs, S. N. (2011). The 

vaccinia virus complement control protein modulates adaptive immune responses during 

infection. J Virol 85, 2547–2556. doi:10.1128/JVI.01474-10 Medline 

Gras, S., Burrows, S. R., Kjer-Nielsen, L., Clements, C. S., Liu, Y. C., Sullivan, L. C., Bell, M. 

J., Brooks, A. G., Purcell, A. W. & McCluskey, J. (2009). The shaping of T cell receptor 

recognition by self-tolerance. Immunity 30, 193–203. doi:10.1016/j.immuni.2008.11.011 

Medline 

Harris, S. L., Frank, I., Yee, A., Cohen, G. H., Eisenberg, R. J. & Friedman, H. M. (1990). 

Glycoprotein C of herpes simplex virus type 1 prevents complement-mediated cell lysis and 

virus neutralization. J Infect Dis 162, 331–337. doi:10.1093/infdis/162.2.331 Medline 

Huang, I. C., Li, W., Sui, J., Marasco, W., Choe, H. & Farzan, M. (2008). Influenza A virus 

neuraminidase limits viral superinfection. J Virol 82, 4834–4843. doi:10.1128/JVI.00079-08 

Medline 

Hutin, Y. J., Williams, R. J., Malfait, P., Pebody, R., Loparev, V. N., Ropp, S. L., Rodriguez, 

M., Knight, J. C., Tshioko, F. K. & other authors (2001). Outbreak of human 

monkeypox, Democratic Republic of Congo, 1996 to 1997. Emerg Infect Dis 7, 434–438. 

Medline 


Ichihashi, Y. (1977). Vaccinia-specific hemagglutinin. Virology 76, 527–538. doi:10.1016/0042- 

6822(77)90235-5 Medline 

Ichihashi, Y. & Dales, S. (1971). Biogenesis of poxviruses: interrelationship between 

hemagglutinin production and polykaryocytosis. Virology 46, 533–543. doi:10.1016/0042- 

6822(71)90057-2 Medline 

Isaacs, S. N., Kotwal, G. J. & Moss, B. (1992). Vaccinia virus complement-control protein 

prevents antibody-dependent complement-enhanced neutralization of infectivity and 

contributes to virulence. Proc Natl Acad Sci U S A 89, 628–632. doi:10.1073/pnas.89.2.628 

Medline 

Izmailyan, R. & Chang, W. (2008). Vaccinia virus WR53.5/F14.5 protein is a new component of 

intracellular mature virus and is important for calcium-independent cell adhesion and 

vaccinia virus virulence in mice. J Virol 82, 10079–10087. doi:10.1128/JVI.00816-08 

Medline 

Jin, D. Y., Li, Z. L., Jin, Q., Hao, Y. W. & Hou, Y. D. (1989). Vaccinia virus hemagglutinin. A 

novel member of the immunoglobulin superfamily. J Exp Med 170, 571–576. 

doi:10.1084/jem.170.2.571 Medline 

Kotwal, G. J. & Moss, B. (1988). Vaccinia virus encodes a secretory polypeptide structurally 

related to complement control proteins. Nature 335, 176–178. doi:10.1038/335176a0 

Medline 

Kotwal, G. J., Isaacs, S. N., McKenzie, R., Frank, M. M. & Moss, B. (1990). Inhibition of the 

complement cascade by the major secretory protein of vaccinia virus. Science 250, 827–830. 

doi:10.1126/science.2237434 Medline 

Law, K. M. & Smith, G. L. (1992). A vaccinia serine protease inhibitor which prevents virus- 

induced cell fusion. J Gen Virol 73, 549–557. doi:10.1099/0022-1317-73-3-549 Medline 

Law, M., Carter, G. C., Roberts, K. L., Hollinshead, M. & Smith, G. L. (2006). Ligand-induced 

and nonfusogenic dissolution of a viral membrane. Proc Natl Acad Sci U S A 103, 5989– 

5994. doi:10.1073/pnas.0601025103 Medline 

Lee, M. S., Roos, J. M., McGuigan, L. C., Smith, K. A., Cormier, N., Cohen, L. K., Roberts, B. 

E. & Payne, L. G. (1992). Molecular attenuation of vaccinia virus: mutant generation and 

animal characterization. J Virol 66, 2617–2630. Medline 

Lin, S. F., Price, D. L., Chen, C. H., Brader, P., Li, S., Gonzalez, L., Zhang, Q., Yu, Y. A., 

Chen, N. & other authors (2008). Oncolytic vaccinia virotherapy of anaplastic thyroid 

cancer in vivo. J Clin Endocrinol Metab 93, 4403–4407. doi:10.1210/jc.2008-0316 Medline 


Lindwasser, O. W., Chaudhuri, R. & Bonifacino, J. S. (2007). Mechanisms of CD4 

downregulation by the Nef and Vpu proteins of primate immunodeficiency viruses. Curr 

Mol Med 7, 171–184. doi:10.2174/156652407780059177 Medline 

Liszewski, M. K., Leung, M. K., Hauhart, R., Buller, R. M., Bertram, P., Wang, X., 

Rosengard, A. M., Kotwal, G. J. & Atkinson, J. P. (2006). Structure and regulatory 

profile of the monkeypox inhibitor of complement: comparison to homologs in vaccinia and 

variola and evidence for dimer formation. J Immunol 176, 3725–3734. Medline 

Liszewski, M. K., Leung, M. K., Hauhart, R., Fang, C. J., Bertram, P. & Atkinson, J. P. 

(2009). Smallpox inhibitor of complement enzymes (SPICE): dissecting functional sites and 

abrogating activity. J Immunol 183, 3150–3159. doi:10.4049/jimmunol.0901366 Medline 

Manes, N. P., Estep, R. D., Mottaz, H. M., Moore, R. J., Clauss, T. R., Monroe, M. E., Du, X., 

Adkins, J. N., Wong, S. W. & Smith, R. D. (2008). Comparative proteomics of human 

monkeypox and vaccinia intracellular mature and extracellular enveloped virions. J 

Proteome Res 7, 960–968. doi:10.1021/pr070432+ Medline 

McKenzie, R., Kotwal, G. J., Moss, B., Hammer, C. H. & Frank, M. M. (1992). Regulation of 

complement activity by vaccinia virus complement-control protein. J Infect Dis 166, 1245– 

1250. doi:10.1093/infdis/166.6.1245 Medline 

Miller, C. G., Shchelkunov, S. N. & Kotwal, G. J. (1997). The cowpox virus-encoded homolog of 

the vaccinia virus complement control protein is an inflammation modulatory protein. 

Virology 229, 126–133. doi:10.1006/viro.1996.8396 Medline 

Moss, B. (2006). Poxvirus entry and membrane fusion. Virology 344, 48–54. 

doi:10.1016/j.virol.2005.09.037 Medline 

Moulton, E. A., Bertram, P., Chen, N., Buller, R. M. & Atkinson, J. P. (2010). Ectromelia virus 

inhibitor of complement enzymes protects intracellular mature virus and infected cells from 

mouse complement. J Virol 84, 9128–9139. doi:10.1128/JVI.02677-09 Medline 

Mullick, J., Bernet, J., Panse, Y., Hallihosur, S., Singh, A. K. & Sahu, A. (2005). Identification 

of complement regulatory domains in vaccinia virus complement control protein. J Virol 79, 

12382–12393. doi:10.1128/JVI.79.19.12382-12393.2005 Medline 

Nagler, F. P. O. (1942). Application of Hirst's phenomenon to the titration of vaccinia virus and 

vaccinia immune serum. Med J Aust 1, 281–283. 

Oda, M. (1965). Rescue of dermovaccinia abortive infection by neurovaccinia virus in L cells. 

Virology 25, 664–666. doi:10.1016/0042-6822(65)90096-6 Medline 


Oie, M., Shida, H. & Ichihashi, Y. (1990). The function of the vaccinia hemagglutinin in the 

proteolytic activation of infectivity. Virology 176, 494–504. doi:10.1016/0042- 

6822(90)90019-N Medline 

Payne, L. G. (1992). Characterization of vaccinia virus glycoproteins by monoclonal antibody 

precipitation. Virology 187, 251–260. doi:10.1016/0042-6822(92)90313-E Medline 

Payne, L. G. & Norrby, E. (1976). Presence of haemagglutinin in the envelope of extracellular 

vaccinia virus particles. J Gen Virol 32, 63–72. doi:10.1099/0022-1317-32-1-63 Medline 

Pruitt, K. D., Tatusova, T. & Maglott, D. R. (2007). NCBI reference sequences (RefSeq): a 

curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic 

Acids Res 35 (Database issue), D61–D65. doi:10.1093/nar/gkl842 Medline 

Pulford, D. J., Gates, A., Bridge, S. H., Robinson, J. H. & Ulaeto, D. (2004). Differential 

efficacy of vaccinia virus envelope proteins administered by DNA immunisation in 

protection of BALB/c mice from a lethal intranasal poxvirus challenge. Vaccine 22, 3358– 

3366. doi:10.1016/j.vaccine.2004.02.034 Medline 

Rosengard, A. M., Alonso, L. C., Korb, L. C., Baldwin, W. M., III, Sanfilippo, F., Turka, L. A. 

& Ahearn, J. M. (1999). Functional characterization of soluble and membrane-bound forms 

of vaccinia virus complement control protein (VCP). Mol Immunol 36, 685–697. 

doi:10.1016/S0161-5890(99)00081-4 Medline 

Rosengard, A. M., Liu, Y., Nie, Z. & Jimenez, R. (2002). Variola virus immune evasion design: 

expression of a highly efficient inhibitor of human complement. Proc Natl Acad Sci U S A 

99, 8808–8813. Medline 

Sahu, A., Isaacs, S. N., Soulika, A. M. & Lambris, J. D. (1998). Interaction of vaccinia virus 

complement control protein with human complement proteins: factor I-mediated 

degradation of C3b to iC3b1 inactivates the alternative complement pathway. J Immunol 

160, 5596–5604. Medline 

Seki, M., Oie, M., Ichihashi, Y. & Shida, H. (1990). Hemadsorption and fusion inhibition 

activities of hemagglutinin analyzed by vaccinia virus mutants. Virology 175, 372–384. 

doi:10.1016/0042-6822(90)90422-N Medline 

Senkevich, T. G., Ojeda, S., Townsley, A., Nelson, G. E. & Moss, B. (2005). Poxvirus 

multiprotein entry-fusion complex. Proc Natl Acad Sci U S A 102, 18572–18577. 

doi:10.1073/pnas.0509239102 Medline 


Sfyroera, G., Katragadda, M., Morikis, D., Isaacs, S. N. & Lambris, J. D. (2005). Electrostatic 

modeling predicts the activities of orthopoxvirus complement control proteins. J Immunol 

174, 2143–2151. Medline 

Shida, H. (1986a). Nucleotide sequence of the vaccinia virus hemagglutinin gene. Virology 150, 

451–462. doi:10.1016/0042-6822(86)90309-0 Medline 

Shida, H. (1986b). Variants of vaccinia virus hemagglutinin altered in intracellular transport. Mol 

Cell Biol 6, 3734–3745. Medline 

Shida, H. (1989). Vaccinia virus hemagglutinin. Subcell Biochem 15, 405–440. Medline 

Shida, H. & Dales, S. (1981). Biogenesis of vaccinia: carbohydrate of the hemagglutinin 

molecules. Virology 111, 56–72. doi:10.1016/0042-6822(81)90653-X Medline 

Shida, H. & Matsumoto, S. (1983). Analysis of the hemagglutinin glycoprotein from mutants of 

vaccinia virus that accumulates on the nuclear envelope. Cell 33, 423–434. 

doi:10.1016/0092-8674(83)90424-5 Medline 

Shida, H., Hinuma, Y., Hatanaka, M., Morita, M., Kidokoro, M., Suzuki, K., Maruyama, T., 

Takahashi-Nishimaki, F., Sugimoto, M. & other authors (1988). Effects and virulences 

of recombinant vaccinia viruses derived from attenuated strains that express the human T- 

cell leukemia virus type I envelope gene. J Virol 62, 4474–4480. Medline 

Simon, K. O., Cardamone, J. J., Jr, Whitaker-Dowling, P. A., Youngner, J. S. & Widnell, C. 

C. (1990). Cellular mechanisms in the superinfection exclusion of vesicular stomatitis virus. 

Virology 177, 375–379. doi:10.1016/0042-6822(90)90494-C Medline 

Spiro, R. G. (1966). Characterization of carbohydrate units of glycoproteins. Methods Enzymol 8, 

26–52. doi:10.1016/0076-6879(66)08006-6 

Thompson, J. P., Turner, P. C., Ali, A. N., Crenshaw, B. C. & Moyer, R. W. (1993). The effects 

of serpin gene mutations on the distinctive pathobiology of cowpox and rabbitpox virus 

following intranasal inoculation of Balb/c mice. Virology 197, 328–338. 

doi:10.1006/viro.1993.1594 Medline 

Turner, P. C. & Moyer, R. W. (1992). An orthopoxvirus serpinlike gene controls the ability of 

infected cells to fuse. J Virol 66, 2076–2085. Medline 

Turner, P. C. & Moyer, R. W. (1995). Orthopoxvirus fusion inhibitor glycoprotein SPI-3 (open 

reading frame K2L) contains motifs characteristic of serine proteinase inhibitors that are not 

required for control of cell fusion. J Virol 69, 5978–5987. Medline 


Turner, P. C. & Moyer, R. W. (2006). The cowpox virus fusion regulator proteins SPI-3 and 

hemagglutinin interact in infected and uninfected cells. Virology 347, 88–99. 

doi:10.1016/j.virol.2005.11.012 Medline 

Turner, P. C. & Moyer, R. W. (2008). The vaccinia virus fusion inhibitor proteins SPI-3 (K2) and 

HA (A56) expressed by infected cells reduce the entry of superinfecting virus. Virology 380, 

226–233. doi:10.1016/j.virol.2008.07.020 Medline 

Turner, P. C., Baquero, M. T., Yuan, S., Thoennes, S. R. & Moyer, R. W. (2000). The cowpox 

virus serpin SPI-3 complexes with and inhibits urokinase-type and tissue-type plasminogen 

activators and plasmin. Virology 272, 267–280. doi:10.1006/viro.2000.0377 Medline 

Wagenaar, T. R. & Moss, B. (2007). Association of vaccinia virus fusion regulatory proteins with 

the multicomponent entry/fusion complex. J Virol 81, 6286–6293. doi:10.1128/JVI.00274- 

07 Medline 

Wagenaar, T. R. & Moss, B. (2009). Expression of the A56 and K2 proteins is sufficient to inhibit 

vaccinia virus entry and cell fusion. J Virol 83, 1546–1554. doi:10.1128/JVI.01684-08 

Medline 

Wagenaar, T. R., Ojeda, S. & Moss, B. (2008). Vaccinia virus A56/K2 fusion regulatory protein 

interacts with the A16 and G9 subunits of the entry fusion complex. J Virol 82, 5153–5160. 


Wang, Y. X., Turner, P. C., Ness, T. L., Moon, K. B., Schoeb, T. R. & Moyer, R. W. (2000). 

The cowpox virus SPI-3 and myxoma virus SERP1 serpins are not functionally 

interchangeable despite their similar proteinase inhibition profiles in vitro. Virology 272, 

281–292. doi:10.1006/viro.2000.0378 Medline 

Wildum, S., Schindler, M., Münch, J. & Kirchhoff, F. (2006). Contribution of Vpu, Env, and 

Nef to CD4 down-modulation and resistance of human immunodeficiency virus type 1- 

infected T cells to superinfection. J Virol 80, 8047–8059. doi:10.1128/JVI.00252-06 

Medline 

Worschech, A., Chen, N., Yu, Y. A., Zhang, Q., Pos, Z., Weibel, S., Raab, V., Sabatino, M., 

Monaco, A. & other authors (2009). Systemic treatment of xenografts with vaccinia virus 

GLV-1h68 reveals the immunologic facet of oncolytic therapy. BMC Genomics 10, 301. 

doi:10.1186/1471-2164-10-301 Medline 

Yu, Y. A., Galanis, C., Woo, Y., Chen, N., Zhang, Q., Fong, Y. & Szalay, A. A. (2009a). 

Regression of human pancreatic tumor xenografts in mice after a single systemic injection 

of recombinant vaccinia virus GLV-1h68. Mol Cancer Ther 8, 141–151. doi:10.1158/1535- 

7163.MCT-08-0533 Medline 


Yu, Z., Li, S., Brader, P., Chen, N., Yu, Y. A., Zhang, Q., Szalay, A. A., Fong, Y. & Wong, R. 

J. (2009b). Oncolytic vaccinia therapy of squamous cell carcinoma. Mol Cancer 8, 45. 

doi:10.1186/1476-4598-8-45 Medline 

Zhou, J., Sun, X. Y., Fernando, G. J. & Frazer, I. H. (1992). The vaccinia virus K2L gene 

encodes a serine protease inhibitor which inhibits cell-cell fusion. Virology 189, 678–686. 

doi:10.1016/0042-6822(92)90591-C Medline 


Fig. 1. Diagram of the location of the A56 protein in a VACV-infected cell. A56, K2 and VCP all 

have signal sequences that result in their being trafficked through the endoplasmic reticulum (ER). 

Presumably, initial protein–protein interactions take place here, followed by movement through the 

Golgi apparatus and to the cytoplasmic membrane. As a transmembrane protein, A56 is found on 

the surface of the infected cell, where it is present as a monomer, as well as in complex with the K2 

protein and with VCP. Since A56 is also one of the glycoproteins found on the extracellular virus 

(EV), the A56–K2 complex (and probably the A56–VCP complex) is also expressed on the EV 

outer membrane. EV is formed by a small portion of MV interacting with the Golgi apparatus, 

where the MV picks up an additional membrane that contains an additional set of membrane 

proteins, including A56. 

Fig. 2. Schematic map of the domains of the A56 protein. After a cleaved signal sequence (aa 1– 

16), there is an Ig domain (aa 17–105), which includes a predicted intramolecular disulfide bridge 

(between cysteines 34 and 103), a stalk region (aa 121–275) containing tandem repeats (aa 170– 

240, striped boxes) that are highly modified by O-linked glycosylation, a transmembrane domain 

(aa 276–303, white box), and a short cytoplasmic tail (aa 304–315, grey box). The Ig domain is 

believed to be involved in binding the K2 protein. Cysteine 162 forms a disulfide bridge with the 

free N-terminal cysteine of VCP. Also shown are the approximate locations of the predicted Nlinked 

glycosylation sites (grey lollipops) at aa 37, 69, 112, 161 and 254. 

Fig. 3. Predicted structure of the N terminus of the A56 protein and homology modelling of the Ig 

domain of A56. Predicted beta sheets are coloured yellow and predicted alpha helices are coloured 

red; random coils are coloured green. The predicted disulfide bridge between cysteine residues 34 

and 102 is indicated by orange spheres. A buried tryptophan residue at aa 29 that is conserved 

among IgG-like domains is coloured blue. Outside of the model of the Ig domain, the unpaired 

cysteine at aa 162 is shown. The IgG-like domain of A56 was used as a query sequence for five 

iterations of PSI–BLAST (Altschul et al., 1997) searching of the National Center for Biotechnology 

Information (NCBI) RefSeq database (Pruitt et al., 2007). A large number of proteins with sequence 

homology were identified; the best scoring protein from this query for which an atomic structure 

has been determined is the T-cell-receptor alpha-chain IgG domain from CF34 [PDB 3FFC, chain 

D (Gras et al., 2009)], with an E score of 9.02×10 �27 and 22% sequence identity. The A56 sequence 

was threaded onto this three-dimensional structure using the homology modelling program 

MODELLER 9v8 (Eswar et al., 2006). The resulting model was then energy minimized by using the 

program CNS version 1.3 (Brünger et al., 1998). The overall quality of this minimized model was 

evaluated using the MOLPROBITY server (Chen et al., 2010). It was found that 98.9% of the residues 

resided in the allowable regions of a Ramachandran plot and had overall root-mean-square 

deviations for bonds and angles of 0.004 and 0.671, respectively. The model was rendered using the 

program PYMOL. 


Table 1. Summary of in vivo work with A56 and/or VCP and K2 mutant viruses 

C, Intracranial; IN, intranasal; ID, intradermal; IP, intraperitoneal; WT, wild type; K2ko, K2L ORF deletion; VCPko, VCP ORF deletion; 

VCPmut, mutated N-terminal cysteine that results in VCP that does not form a homodimer and does interact with A56. The relative degree 

of attenuation observed is indicated by the number of plus (+) signs. 

Virus strain or gene mutation 

A56-knockout viruses 

Route Attenuation Notes References 

NYCBH IC ++ LD50=1.6×10 5 p.f.u. versus 1.9×10 2 NYCBH IN +++ 

p.f.u. for WT 

LD50 >1×10 

Lee et al. (1992) 

8 p.f.u. versus 2.5×10 4 WR IC + 

p.f.u. for WT 

100% death at 17 days; 100% death for WT was 8 days 

Lee et al. (1992) 

Flexner et al. (1987) 

WR IP ++ LD50=7.8×10 7 p.f.u. versus 9.3×10 5 p.f.u. for WT (A56R ORF was replaced with 

HTLV glycoprotein) 

Shida et al. (1988) 

WR IC ++ LD50=1.5×10 2 p.f.u. versus

A56/K2 

A56 

A56/VCP 

ER/secretory 

pathway Viral factory 

EV 

MV 

Nucleus

NH2- 

34 

162 - 

170 - 

240 - 

275 - 

304 - 

COOH - 

—C—C— 

103 

120 - 

-C 

Ig Domain 

C103Y mutant – HA negative; syncytia forming 

E121K mutant – HA negative only 

VCP-binding cysteine 

Tandem repeats 

Transmembrane region 

Intracellular domain

C103 

N 

C162 

C 

C34

Page 1 of 22 The vaccinia virus A56 protein: a multifunctional ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?